Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) and is elevated with several risk factors for cardiovascular disease. We have previously demonstrated a direct relationship between the degree of insulin resistance, a cardinal feature of obesity, and circulating levels of ADMA. Our preliminary data indicate that adipose tissue elaborates significant quantities of ADMA. Moreover, this effect is accentuated in tissue derived from obese animals. We hypothesize that adipose tissue can serve as a major source of ADMA and that insulin resistance has important modulatory functions. Thus we will use our in vitro and in vivo models to elucidate the following specific aims: 1. To identify the cellular source(s) of ADMA elaboration from adipose tissue and potential regulatory mechanisms. We will isolate adipocytes and stromal endothelial cells from fat pads to determine the relative contribution of each to ADMA production. In addition, mechanisms of ADMA metabolism will be elucidated in these cells. 2. To determine the role of insulin resistance in adipose tissue ADMA production. Insulin resistance will be induced both in vivo and in vitro and the resultant effects upon ADMA regulation will be assessed. Dysregulation of candidate signaling molecules upon ADMA will be determined as well as the potential benefits of insulin sensitizing compounds (TZDs). 3. To modulate ADMA metabolism by overexpression of DDAH and determine the effects upon ADMA elaboration and cell function. Adipocytes and endothelial cells stably overexpressing DDAH will be examined for functional consequences. In addition, a transplant model will be used to examine the potential therapeutic benefit of ectopic DDAH expression. 4. To examine the expression profile of cardiovascular tissues derived from animals that have altered ADMA metabolism. We will utilize a comprehensive cDNA microarray to examine the effects of obesity, insulin resistance, and ADMA metabolism in cardiovascular tissues. Expression profiles will also be compared with non-cardiovascular tissues. Understanding the questions underlying these specific aims will add insight into the underlying mechanisms of ADMA regulation and endothelial dysfunction and undoubtedly have important implications for the treatment of obesity-related cardiovascular disease.